Project Summary During meiosis the faithful inheritance of the genome is necessary for successful gamete formation. While many tissues are affected by extreme temperature changes, developing sperm in the testes are particularly sensitive to small fluctuations in temperature, with spermatogenesis requiring a narrow isotherm of 2-7C below core body temperature. Testes exposed to high temperature display reduced fertility. Studies in mammals have linked elevated temperatures with an increase in DNA damage in spermatocytes, however the underlying mechanisms remain unknown. Previous work from the Libuda lab found that, similar to mammals, exposure to heat-stress produces DNA damage specifically in Caenorhabditis elegans spermatocytes and not oocytes. Utilizing C. elegans as a model system, transposon mobilization was identified as a possible mechanism underlying the production of heat-induced DNA damage. Small non-coding RNAs, in complex with associated proteins, are crucial regulators of germ line development and maintenance, including the regulation of RNAi and transposon activity. Certain small RNA pathways are also known to be spermatocyte-specific and play a role in temperature-induced infertility. As such, small RNA pathways in the germ line represent a promising target as regulators of heat-stress induced DNA damage in spermatocytes. Therefore, I hypothesize that heat-stress induced DNA damage specifically in spermatocytes is due to transposon mobilization which is regulated by small RNA pathways in the germ line. To test this, I will take a multipronged approach, combining a candidate mutant approach with unbiased RNA sequencing to identify components involved in temperature-induced DNA damage. In Aim 1, I will complete my candidate mutant screen, monitoring temperature-induced DNA damage in small RNA pathway mutants. I will also use RNA sequencing to characterize all temperature-sensitive small RNA populations in an unbiased manner. In Aim 2, I will follow up on my finding that PRG-1, which interacts with piRNAs in the germ line to suppress transposons, is required for heat-stress induced DNA damage. I will investigate my hypothesis that PRG-1 regulates specific piRNA subclasses that mediate the production of temperature-induced DNA damage in spermatocytes with a small RNA sequencing experiment optimized for piRNA analysis. To further explore this result, I will characterize heat shock-dependent localization and interactions of PRG-1, associated piRNAs, and additional small RNA pathway components known to act downstream of PRG-1. In Aim 3, I will assess transposon mobilization upon heat-shock and characterize transposon classes involved in heat-stress induced DNA damage. I propose to combine deep sequencing and genetic approaches to explore how temperature-induced DNA damage occurs specifically in spermatocytes using the nematode C. elegans. Overall, these data will make a substantial contribution toward improving our understanding of these important biological processes that are relevant to human infertility and disease.